PROJECT SUMMARY The objective of this Fellowship is to define the contribution of Endomucin (EMCN) to angiogenesis, with an emphasis on defining its regulation of tyrosine kinase receptors, namely vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2), through its extracellular domain. In vitro studies confirmed that EMCN altered VEGF-induced proliferation, migration and tube formation, by modulating VEGFR2 phosphorylation and internalization. However, EMCN did not directly interact with VEGFR2. Interestingly, EMCN showed a direct interaction with galectin-3 (Gal3), and co-localized with Gal3 and VEGFR2 on the endothelial cell (EC) surface. Loss of EMCN was sufficient to prevent both VEGF- and Gal3-induced angiogenesis. This proposal will test the hypothesis that EMCN, Gal3 and VEGFR2 interact on EC surface, in a carbohydrate-dependent manner, and that crosstalk between these factors mediates VEGFR2 signaling. Aim 1 will define the role of EMCN in regulating VEGFR2 ligand occupancy, signal transduction and trafficking, with an emphasis on identifying the contribution of the extracellular and intracellular domains. This will be investigated using CRISPR/cas9 technology to edit the genomic locus of EMCN in vitro, in the presence or absence of truncated EMCN mutants. Aim 2 will define the contribution of EMCN and Gal3 interactions, and their role in modulating VEGF-induced angiogenesis. Specifically, the role of EMCN?s post-translational modifications (i.e. N- or O-glycosylation), will be characterized using small molecular inhibitors. Due to EMCN?s exclusive localization in ECs, targeting EMCN may represent a unique opportunity for specificity that is not provided by pan-VEGF neutralization. Aim 3 will evaluate the effect of EMCN knockdown, using targeted siRNA, in combination with anti-VEGF (Aflibercept), on preventing neovascularization in a mouse model of laser-induced choroid neovascularization. This project is significant in that it will define a role for EMCN in regulating optimal VEGFR2 activity, through cell surface interactions and define a therapeutic role for EMCN, as an EC-specific target. The long-term goal of this Fellowship is to define the contribution of post-translational modifications in modulating EMCN cell surface interactions, with therapeutic potential.